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Structural identification of lipopeptide biosurfactants produced by Bacillus subtilis strains grown on the media obtained from renewable natural resources
Journal of Environmental Management 209 (2018) 65e70
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Journal of Environmental Management journal homepage: www.elsevier.com/locate/jenvman
Research article
Structural identification of lipopeptide biosurfactants produced by Bacillus subtilis strains grown on the media obtained from renewable natural resources Katarzyna Paraszkiewicz a, Przemysław Bernat a, Anna Kusmierska a, Joanna Chojniak b, _ Grazyna Płaza b, * a Department of Industrial Microbiology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha Street 12/16, 90dz, Poland 237, Ło b Institute for Ecology of Industrial Areas, Environmental Microbiology Unit, Kossutha Street 6, 40-844, Katowice, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2017 Received in revised form 12 December 2017 Accepted 14 December 2017
The aim of the study was to identify and characterize lipopeptide (LP) biosurfactants produced by two Bacillus subtilis strains (KP7 and I0 -1a) grown on various media prepared from renewable natural resources: two different brewery wastewaters (BW#4 and BW#6), 2% beet molasses (M), apple peels extract (APE) supplemented with 0.25% of yeast extract (YE) or 0.25% peptone (P), and similarly supplemented carrot peels extract (CPE). In all used media both strains retained their individual LP production signature characterized by surfactin and iturin overproduction exhibited by KP7 and I0 -1a strain, respectively. The production level and the structural diversity of synthesized LPs were dependent on the medium composition. In the CPEþYE medium it was higher than the yield obtained in Luria-Bertani (140.6 and 100.3 mg L1, respectively). Surfactins were produced by both strains as a mixture of four homologues (C13-C16) with the domination of variant C14. All other broths prepared from renewable resources strongly stimulated the iturin production by I0 -1a strain with the exception of BW media. The highest iturin concentration (428.7 mg L1) obtained in the CPEþP culture of I0 -1a strain was about seven-fold higher than in LB. In all cultures only iturin A was identified. Among four iturin homologues (C13-16) produced by I0 -1a strain, the highest relative contents of C16 variant (70e80%) were calculated for samples obtained from APEþP and CPEþP media. The obtained data indicate that the waste composition has an influence on both the types and amounts of biosurfactants produced by studied B. subtilis strains. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Agro-industrial wastes Bacillus spp. Biosurfactants Surfactin Iturin Renewable natural resources
1. Introduction Now, several industries have been increasingly directed towards biotechnology and circular bioeconomy (Communication EU, 13.2.2012 COM, 2012). Worldwide interest in biosurfactants(considered as “green technology” products)has significantly grown in recent years due to their more environmentally friendly properties compared to the chemical surfactants (Paraszkiewicz, 2016). The worldwide biosurfactant production is expected to reach 476,512.2 tons by 2018 with the global surfactant market worth more than US $41 billion (Kapadia and Yagnik, 2013;
* Corresponding author. E-mail address: [email protected] (G. Płaza). https://doi.org/10.1016/j.jenvman.2017.12.033 0301-4797/© 2017 Elsevier Ltd. All rights reserved.
Dhanarajan and Sen, 2015). Commercial biosurfactant production is discussed frequently in the literature, debating the large difference in the monetary inputs versus actual financial gain. The development of cheaper processes of biosurfactant production and the use of low-cost raw material are important factors that account for 10e30% of the overall cost (Kosaric and Vardar-Sukan, 2015). Agroindustrial wastes are considered as a promising substrate for microbial synthesis of biosurfactants and can solve various industrial waste management problems (Makkar et al., 2011). Consequently, research has focused on the usage of several effective renewable natural resources to overcome the financial hurdles in the biosurfactant production industry. Currently, substrates such as wheat straw, rice straw, cassava, cassava flour, sugarcane molasses, bagasse of sugarcane, beet molasses, bran, and corn are being tested for biosurfactant production at the commercial level (Banat
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et al., 2014; Mnif et al., 2012). Satpute et al. (2017) discuss a novel and efficient way of using agro-industrial, dairy and food processing waste for biosurfactant production. It is very important to recycle and reuse various renewable substrates as ingredients to recover industrial-scale products (Kosaric and Vardar-Sukan, 2015). At the same time, it is very important to choose top-quality substrates in terms of nutritional value that can allow the growth of desired microorganisms along with plentiful production of microbial surface active agents. Bacillus subtilis is a well-studied producer of a variety of cyclic LP biosurfactants from which the best characterized are compounds from surfactin, iturin and fengycin families. Bacilli LPs exhibit interesting physiological, biocidal, physicochemical, and surfaceactive properties (Raaijmakers et al., 2010; Wang et al., 2015; Diaz De Rienzo et al., 2016). Surfactins are mostly used as emulsifying and foaming agents in remediation technologies and iturin derivatives display a strong in vitro and in vivo antimicrobial activity against fungi belonging to various families. Due to their natural diversity, LPs are synthesized by particular strains as mixtures of isoforms and homologues differing in the amino acid composition of the peptide sequence and in the length of the fatty acid chain, respectively (Pecci et al., 2010). Studying the relationships between yields and molecular structures of synthesized LPs and the kind of wastes/by-products used as growth media components is considered to be important for the development of cost effective technologies enabling the production of novel biochemicals with different properties and their potential use in industrial applications. In this context, the above paper addresses the potential of various agro-industrial wastes for surfactin and iturin production. Moreover, structural diversity of the LP biosurfactants produced by two studied B. subtilis strains has been determined.
2. Materials
cultures obtained from LB medium and adjusted to OD600nm 0.9; (approximately 107e108 CFU mL) were introduced as a 2% inoculum to 300 mL Erlenmeyer flasks containing 150 mL of the selected medium. The cultures were grown aerobically for 96 h with constant shaking (120 rpm) at 28 C.The following media were used to evaluate LPs production: Luria-Bertani (LB) broth; Cooper's broth (Al-Ajlani et al., 2007), two different brewery wastewaters (BW) obtained from beer production based on barley malt and wheat malt (signed BW#4 and BW#6, respectively), 2% beet molasses (M), apple peels extract (APE) supplemented with 0.25% of yeast extract (YE) or peptone (P) and the carrot peels extract (CPE) with the same supplements. Supernatants obtained by centrifugation (10.000g, 20 min) of 96 h cultures were used for LPs isolation by the method based on the QuEChERS technique (Siewiera et al., 2015; Paraszkiewicz et al., 2017). To obtain the vegetable extracts, proper quantities of carrot and apple peels were boiled for 30 min. After centrifugation, the remaining solid particles were removed by filtration. Distilled water was added to the extracts to a final volume of 1000 ml. All media (with pH adjusted to 7) were sterilized before being used. 2.2. Lipopeptides isolation and mass spectrometry analysis Supernatants obtained by centrifugation (10.000g, 20 min) of 96 h cultures were used for LPs isolation according to the method described by Płaza et al. (2015). LP concentrations in culture supernatants as well as their structures were determined by liquid chromatography e mass spectrometry (LC-MS/MS) and matrixassisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF) techniques (Bernat et al., 2016). Surfactin and iturin standards were obtained from Sigma-Aldrich (Germany) and all other chemicals were purchased from Avantor Performance Materials Poland S.A., Sigma-Aldrich (Germany), Serva (Germany) or Fluka (Germany).
2.1. Culturing of Bacillus strains on different media
2.3. Statistical analysis
The research aim, objectives, and methods are briefly described in Table 1. In detail, two strains of Bacillus subtilis named KP7 and I0 1a were used in this study. KP7 strain was obtained from the collection of the Department of Industrial Microbiology and d Biotechnology, University of Ło z, and I0 -1astrain was supplied by the Institute for Ecology of Industrial Areas in Katowice, Poland. Taxonomic identification and preliminary characterization of strains KP7 and I0 -1a have been described previously by Płaza et al. (2015). The strains were preserved at 70 C in LuriaeBertani (LB) medium supplemented with 20% (v/v) glycerol. Bacterial 24-h-old
All the experiments were performed in triplicates and analysed individually. An average standard deviation was calculated. 3. Results and discussion Data presented in Table 2 reveal that in all growth media used in the study B. subtilis strains were able to synthesise both surfactin and iturin. Moreover, regardless of the used growth medium, KP7 strain produced more surfactin than iturin, while I0 -1a strain overproduced iturin with only traces of surfactin. The same, in all
Table 1 Research aim, objectives and methods of the study. Research principle
Description
Research aim
Determination of the potential of various agro-industrial wastes for surfactin and iturin production. Determination of the structural diversity of the biosurfactants produced by two B. subtilis strains cultured in standard and low-cost media KP7 - surfactin overproducer, producing in LB medium surfactin and iturin in the ratio 17.6: 1; I0 -1a e iturin overproducer, producing in LB medium surfactin and iturin in the ratio 1: 19.8 Two different brewery wastewaters (BW) obtained from beer production based on barley malt and wheat malt (signed BW#4 and BW#6, respectively); 2% beet molasses (signed M); the apple peels extract (APE) and the carrot peels extract (CPE) supplemented with 0.25% of yeast extract (YE) or peptone (P)
Research objectives
Methods
a b
Bacillus subtilis strains Cost-effective culture media containing agroindustrial wastes Control (standard) growth media Biosurfactant isolation Biosurfactant analysis
LuriaeBertani (LB) medium and Cooper's medium Modified QuEChERS technique LC-MS/MSa MALDI-TOF/TOFb
Liquid chromatography e mass spectrometry. Matrix-assisted laser desorption time-of-flight mass spectrometry.
K. Paraszkiewicz et al. / Journal of Environmental Management 209 (2018) 65e70 Table 2 Surfactin production by B. subtilis strains. Growth media Standard media Media prepared from industrial wastewaters Media prepared from vegetable extracts
LB Cooper's BW (#4) BW (#6) M APEþ YE APEþ P CPEþ YE CPEþ P
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Table 4 The surfactin/iturin ratio for tested B. subtilis strains. B. subtilis KP7
B. subtilis I0 -1a
Growth media
100.3 ± 9.18 205.4 ± 21.39 6.6 ± 0.69 9.9 ± 9.18 77.9 ± 0.87 95.2 ± 7.47 104.4 ± 12.6 140.6 ± 8.45 123.6 ± 9.1
3.7 ± 0.32 7.5 ± 0.69 2.3 ± 0.21 1.8 ± 0.19 10.4 ± 1.32 6.3 ± 0.64 7.6 ± 0.69 7.5 ± 0.84 13.9 ± 10.2
Standard media Media prepared from industrial wastewaters Media prepared from vegetable extracts
LB Cooper's BW (#4) BW (#6) M APEþ YE APEþ P CPEþ YE CPEþ P
B. subtilis KP7
B. subtilis I0 -1a
(17.6): (16.6): (16.5): (16.5): (21.6): (52.9): (30.7): (43.9): (49.4):
(1): (1): (1): (1): (1): (1): (1): (1): (1):
(1) (1) (1) (1) (1) (1) (1) (1) (1)
(19.8) (3.1) (6.4) (19.7) (10.9) (27.2) (35.4) (30.1) (30.8)
Luria-Bertani (LB) medium; brewery wastewaters from beer production based on barley malt (BW#4) and wheat malt (BW#6); 2% beet molasses (M); apple peels extract (APE); carrot peels extract (CPE); 0.25% of yeast extract (YE); 0.25% peptone (P).
tested media both surfactin and iturin co-producers retained their individual LP production signature. The literature presents that the medium composition is believed to be an important factor affecting both the production level and the structural diversity of synthesized LPs (Al-Ajlani et al., 2007; Li et al., 2008; Bacon et al., 2012). The above findings are in agreement with data presented in this report. Surfactin production calculated for samples of KP7 strain varied from 6.6 to 205.4 mg L1 (Table 2) while the secretion of iturin by I0 -1a strain was in the range 16.6e428.7 mg L1 (Table 3). The highest surfactin concentration (205.4 mg L1) was obtained in the culture of KP7 strain conducted in Cooper's medium. However, in medium prepared from carrot peels with 0.5% yeast extract (CPEþYE) surfactin production achieved by KP7 strain was higher than in LB medium (140.6 mg L1 and 100.3 mg L1, respectively). The most effective media for iturin production by the strain I0 -1a were CPE and APE supplemented with peptone (428.7 and 269.5 mg L1, respectively). Interestingly, none of the tested media increased the production of iturin by KP7 strain. Some reports published recently demonstrated that various bacilli strains when grown on media prepared from agro-industrial wastes were capable of efficient surfactin production. For example, CagriMehmetoglu et al. (2012) demonstrated that B. subtilis ATCC 6633 produced from 180 to 290 mg L1 when grown in media prepared from rehydrated whey powder. In medium containing glycerol as a main carbon source the production of surfactin by B. subtilis LAMI 009 and LAMI 005 reached 441.1 and 267.6 mg L1, respectively (Sousa et al., 2012). Ali et al. (2014) reported that strain Bacillus RMB7 (exhibiting plant growth promoting potential) produced iturin A in LB culture up to 25 mg L1. Kumar et al. (2015) established that sunflower oil cake favoured high iturin A secretion by rhizospheric isolate of B. amyloliquefaciens up to 276 mg L1. The above mentioned data might suggest that used in our study B. subtilis I0 -1a could be considered as an efficient iturin producer. The surfactin/iturin ratio calculated for KP7 samples (surfactin overproducer) has increased approximately 3-fold in cultures carried out on media prepared from vegetable extracts (all variants of APE and CPE media) as compared with the data obtained from all other cultures (Table 4). Interestingly, a similar relationship was
Table 3 Iturin production by B. subtilis strains. Growth media Standard media Media prepared from industrial wastewaters Media prepared from vegetable extracts
LB Cooper's BW (#4) BW (#6) M APEþ YE APEþ P CPEþ YE CPEþ P
B. subtilis KP7
B. subtilis I0 -1a
5.7 ± 0.43 12.4 ± 2.0 0.4 ± 0.03 0.6 ± 0.06 3.6 ± 0.3 1.8 ± 0.24 3.5 ± 0.17 3.2 ± 0.4 2.5 ± 0.28
73.3 ± 5.86 23.3 ± 1.95 14.6 ± 1.32 35.5 ± 3.11 113.1 ± 9.89 171.2 ± 19.75 269.5 ± 23.34 225.9 ± 25.3 428.7 ± 43.8
observed in samples of I0 -1a strain: in all media based on vegetable extracts cells of B. subtilis I0 -1a (iturin overproducer) increased the production of iturin which resulted in the significant growth of the iturin/surfactin ratio (Table 3). The above data might have suggested that some components of the used vegetable extracts could stimulate the secretion of the LP biosurfactant which tends to be overproduced. Qualitative analysis by MALDI TOF/TOF revealed that KP7 produced two isoforms of surfactin. Fragment ions at m/z 1030 / 917/804 / 689/590 revealed the loss of Leu-Leu-AspVal from the C terminus (Fig. 1S). Furthermore, another typical set of yþ fragment ions at m/z 707 / 594/481 / 382/267 suggested the loss of the Leu-Leu-Val-Asp in the middle of the peptide chain. Therefore the main surfactin isoform (synthesized by both strains in all studied media) was surfactin possessing circular GlueLeueLeu-Val-Asp-Leu-Leu peptide and the b-OH fatty acid. Interestingly, when KP7 strain was growing in the media prepared from vegetable extracts also surfactin isoform with substitution of Leu to Val at position 7 of the circular peptide was produced. Four homologues of surfactin (from C13 to C16; sodiated molecules m/ z1016, 1030, 1044 and 1058 were found in the examined cultures (Fig. 2AS) with the domination of variant C14 ranging between 5565% and 70e85% in samples of KP7 and I0 -1a strains, respectively (Fig. 1). Most often surfactins are synthetized as mixtures of three or four homoloques with acyl chain length in the range C13 - C15 or C13 e C16 (Pecci et al., 2010; Płaza et al., 2015). The MALDI-TOF/TOF MS of extracts of I0 -1a strain showed dominated set of ions m/z 1051.5, 1065.6, 1079.5 and 1093.5 (Fig. 2BS). The molecules differed in their masses by 14 suggesting them to be a members of the same family. Two additional sets of m/ z ions (with corresponding mass difference of M-22 and Mþ39 Da) were also detected which were putatively assigned as iturins (m/z 1029.5, 1043.5 and 1057.5 and 1071.5) and their potassium adducts (m/z 1067.5, 1081.5, 1095.5 and 1109.5). The appearance of sodium and potassium adducts along with their protonated species is a common feature in mass spectral studies of lipopeptides (Pathak and Keharia, 2014). According to the obtained results, it seems that the dominated ions were sodium adducts. Each of these adducts was subjected to the fragment ions analysis. The lower-mass region of the MS/MS spectra indicated peaks corresponding to the immonium ions of the individual constituent amino acids, namely Ser (m/z 60), Pro (m/z 70), Gln (m/z 84), Asn (m/z 87) and Tyr (m/z 136). Thus, the linear acylium ions of iturin could be Pro-Asn-SerbAA-Asn-Tyr-Asn-Gln-COþ. Taken together, these results confirmed that the compound belongs to Iturin A family. MALDI TOF/TOF analysis revealed that both strains produced iturin A. Among four iturin homologues (C13-16) produced by I0 -1a strain, the highest relative contents of C16 variant (70e80%) were calculated for samples obtained from APEþP and CPEþP media (Fig. 2).
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Fig. 1. Relative contents of surfactin homologes produced by B. subtilis KP7 and I0 -1a strains in media prepared from apple peels extract (APE) and carrot peels extract (CPE) supplemented with 0.25% of yeast extract (YE) or 0.25% of peptone (P). Due to a very low abundance (below 3%) the homologue C16 has been not included in the presented calculation.
Fig. 2. Relative contents of iturin A homologes produced by B. subtilis I0 -1a strain in media prepared from apple peels extract (APE) and carrot peels extract (CPE) supplemented with 0.25% of yeast extract (YE) or 0.25% of peptone (P).
On the basis of amino acids variation in the peptide moiety, iturin isomers are classified as iturins A, C, D or E). Moreover iturin A belongs to one of the most frequently detected iturin analogs (Pathak and Keharia, 2014; Cochrane and Vederas, 2014). With the exception of iturin C which does not exhibit the antibiotic activity, other iturin isomers reveal increasing biological activity with a growing number of carbon atoms in the fatty acid chain. In conclusion, the data obtained in this research revealed a great potential of I0 -1a strain due to its ability for very effective production of iturin A during the growth on media prepared from vegetable extracts.
The potential applications of biosurfactants in various industries has significantly increased in recent years; however, economical large-scale production of biosurfactants remains a challenge (Dubey et al., 2012). Preliminary work by Płaza et al. (2011) confirmed that three Bacillus spp. were capable of growing and producing biosurfactants in media composed of brewery effluents, molasses and fruit as well as vegetable decoction from a processing factory. More recently, simultaneous production of detergent stable enzymes and biosurfactant by Bacillus subtilis PF1 growing in the single medium containing cost effective agroindustrial wastes (namely feather meal, potato peel and rape seed cake) was reported
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~ ez-Ocampo et al. (2017) used waste by Bhangea et al. (2016). Yan cooking oil and coffee wastewater as carbon sources for the production of biosurfactants exhibiting the ability to be potentially exploited for the treatment of soil contaminated with insoluble compounds (pesticides or oil hydrocarbons). Various unconventional agroindustrial wastes, (molasses, orange peels extract, bagasse extract, banana peels extract and potato peels extract) were used for the production of biosurfactant from the indigenously isolated Bacillus subtilis ANR 88, examined further towards its possible application in nanoparticle synthesis (Rane et al., 2017). Previously, B. subtilis I0 -1a and KP7 grown on LB medium were described as producers of surfactin, iturin and fengycin mixture (Płaza et al., 2015). Nevertheless, fengycin production by I0 -1a and KP7 strains still requires further study. It was also found that LPs extracted from bacterial cultures had a strong antimicrobial effect on uropathogenic bacteria, including effects on planktonic growth, and biofilm formation and removal (Moryl et al., 2015). Moreover, Bernat et al. (2016) elucidated the structure of LPs produced by B. subtilis strain I0 -1a grown on the traditional microbiological LuriaeBertani (LB) medium. B. subtilis strain I0 -1a was also found to produce a mixture of surfactin homologues (C13-C16). In addition, two iturin A homologues, C14 and C15 were observed, of which C15 was dominant. The obtained results reveal that available renewable natural resources could be used for effective iturin A production by the strain B. subtilis I0 -1a. 4. Conclusions The wastes from various sectors of agro-industry containing many organic compounds could be utilized as substrates for biosurfactant production, which resulted in a double benefit i. e., reduction of the wastes as well as production of valuable residues like biosurfactant. As estimated the use of low-cost raw material is a factor that accounts for a 10e30% of the overall cost of biosurfactant production and can solve various industrial waste management problems. The promising future of biosurfactants depends on the use of a large and low cost raw materials to achieve high biosurfactants yields. In this context, our paper addressed the application of various agro-industrial wastes for biosurfactant production and for the determination of their chemical structure. In detail, the potential of agro-industrial wastes (two different brewery wastewaters, 2% beet molasses, apple peels extract supplemented with 0.25% of yeast extract or 0.25% peptone, and similarly supplemented carrot peels extract) as low-cost culture media substrates for biosurfactant production by two B. subtilis strains was investigated. As compared to data obtained for Luria-Bertani broth (a standard medium used as a control) the production of surfactin by surfactin overproducer B. subtilis KP7 grown in carrot peels extract supplemented with east extract was 140% over the control, whereas the production of iturin by iturin overproducer B. subtilis I0 -1a cultured in carrot peels extract supplemented with peptone was as high as 585% over the control produced. We found that agricultural substrates with different compositions used as bacterial growth media influence the type of biosurfactant produced. The application of wastes in biosurfactant production is still at an early stage. However, the results published in this area are promising. Conflict of interest The authors declare no conflict of interest. Acknowledgements This paper was prepared in connection with work conducted under project (no 2013/09/B/NZ9/01759) that was sponsored by the
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circular bioeconomy. Manag. Sys. Prod. Eng. 1, 46e54. zalska, _ ski, J., 2015. Estradiol improves tributyltin Siewiera, P., Bernat, P., Ro S., Długon degradation by the filamentous fungus Metarhizium robertsii. Int. Biodeter. Biodeg. 104, 258e263. Sousa, M., Melo, V.M., Rodrigues, S., Santana, H.B., Gonçalves, L.R., 2012. Screening of biosurfactant-producing Bacillus strains using glycerol from the biodiesel synthesis as main carbon source. Biop. Biosyst. Eng. 35, 897e906. Wang, T., Liang, Y., Wu, M., Chen, Z., Lin, J., Yang, L., 2015. Natural products from Bacillus subtilis with antimicrobial properties. Chin. J. Chem. Eng. 23, 744e754. ~ ez-Ocampo, G., Somoza-Coutin ~ o, G., Blanco-Gonza lez, C., Wong-Villarreal, A., Yan 2017. Utilization of agroindustrial waste for biosurfactant production by native bacteria from Chiapas. Open Agric. 2, 341e349.
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Research article
Structural identification of lipopeptide biosurfactants produced by Bacillus subtilis strains grown on the media obtained from renewable natural resources Katarzyna Paraszkiewicz a, Przemysław Bernat a, Anna Kusmierska a, Joanna Chojniak b, _ Grazyna Płaza b, * a Department of Industrial Microbiology and Biotechnology, Faculty of Biology and Environmental Protection, University of Lodz, Banacha Street 12/16, 90dz, Poland 237, Ło b Institute for Ecology of Industrial Areas, Environmental Microbiology Unit, Kossutha Street 6, 40-844, Katowice, Poland
a r t i c l e i n f o
a b s t r a c t
Article history: Received 12 January 2017 Received in revised form 12 December 2017 Accepted 14 December 2017
The aim of the study was to identify and characterize lipopeptide (LP) biosurfactants produced by two Bacillus subtilis strains (KP7 and I0 -1a) grown on various media prepared from renewable natural resources: two different brewery wastewaters (BW#4 and BW#6), 2% beet molasses (M), apple peels extract (APE) supplemented with 0.25% of yeast extract (YE) or 0.25% peptone (P), and similarly supplemented carrot peels extract (CPE). In all used media both strains retained their individual LP production signature characterized by surfactin and iturin overproduction exhibited by KP7 and I0 -1a strain, respectively. The production level and the structural diversity of synthesized LPs were dependent on the medium composition. In the CPEþYE medium it was higher than the yield obtained in Luria-Bertani (140.6 and 100.3 mg L1, respectively). Surfactins were produced by both strains as a mixture of four homologues (C13-C16) with the domination of variant C14. All other broths prepared from renewable resources strongly stimulated the iturin production by I0 -1a strain with the exception of BW media. The highest iturin concentration (428.7 mg L1) obtained in the CPEþP culture of I0 -1a strain was about seven-fold higher than in LB. In all cultures only iturin A was identified. Among four iturin homologues (C13-16) produced by I0 -1a strain, the highest relative contents of C16 variant (70e80%) were calculated for samples obtained from APEþP and CPEþP media. The obtained data indicate that the waste composition has an influence on both the types and amounts of biosurfactants produced by studied B. subtilis strains. © 2017 Elsevier Ltd. All rights reserved.
Keywords: Agro-industrial wastes Bacillus spp. Biosurfactants Surfactin Iturin Renewable natural resources
1. Introduction Now, several industries have been increasingly directed towards biotechnology and circular bioeconomy (Communication EU, 13.2.2012 COM, 2012). Worldwide interest in biosurfactants(considered as “green technology” products)has significantly grown in recent years due to their more environmentally friendly properties compared to the chemical surfactants (Paraszkiewicz, 2016). The worldwide biosurfactant production is expected to reach 476,512.2 tons by 2018 with the global surfactant market worth more than US $41 billion (Kapadia and Yagnik, 2013;
* Corresponding author. E-mail address: [email protected] (G. Płaza). https://doi.org/10.1016/j.jenvman.2017.12.033 0301-4797/© 2017 Elsevier Ltd. All rights reserved.
Dhanarajan and Sen, 2015). Commercial biosurfactant production is discussed frequently in the literature, debating the large difference in the monetary inputs versus actual financial gain. The development of cheaper processes of biosurfactant production and the use of low-cost raw material are important factors that account for 10e30% of the overall cost (Kosaric and Vardar-Sukan, 2015). Agroindustrial wastes are considered as a promising substrate for microbial synthesis of biosurfactants and can solve various industrial waste management problems (Makkar et al., 2011). Consequently, research has focused on the usage of several effective renewable natural resources to overcome the financial hurdles in the biosurfactant production industry. Currently, substrates such as wheat straw, rice straw, cassava, cassava flour, sugarcane molasses, bagasse of sugarcane, beet molasses, bran, and corn are being tested for biosurfactant production at the commercial level (Banat
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et al., 2014; Mnif et al., 2012). Satpute et al. (2017) discuss a novel and efficient way of using agro-industrial, dairy and food processing waste for biosurfactant production. It is very important to recycle and reuse various renewable substrates as ingredients to recover industrial-scale products (Kosaric and Vardar-Sukan, 2015). At the same time, it is very important to choose top-quality substrates in terms of nutritional value that can allow the growth of desired microorganisms along with plentiful production of microbial surface active agents. Bacillus subtilis is a well-studied producer of a variety of cyclic LP biosurfactants from which the best characterized are compounds from surfactin, iturin and fengycin families. Bacilli LPs exhibit interesting physiological, biocidal, physicochemical, and surfaceactive properties (Raaijmakers et al., 2010; Wang et al., 2015; Diaz De Rienzo et al., 2016). Surfactins are mostly used as emulsifying and foaming agents in remediation technologies and iturin derivatives display a strong in vitro and in vivo antimicrobial activity against fungi belonging to various families. Due to their natural diversity, LPs are synthesized by particular strains as mixtures of isoforms and homologues differing in the amino acid composition of the peptide sequence and in the length of the fatty acid chain, respectively (Pecci et al., 2010). Studying the relationships between yields and molecular structures of synthesized LPs and the kind of wastes/by-products used as growth media components is considered to be important for the development of cost effective technologies enabling the production of novel biochemicals with different properties and their potential use in industrial applications. In this context, the above paper addresses the potential of various agro-industrial wastes for surfactin and iturin production. Moreover, structural diversity of the LP biosurfactants produced by two studied B. subtilis strains has been determined.
2. Materials
cultures obtained from LB medium and adjusted to OD600nm 0.9; (approximately 107e108 CFU mL) were introduced as a 2% inoculum to 300 mL Erlenmeyer flasks containing 150 mL of the selected medium. The cultures were grown aerobically for 96 h with constant shaking (120 rpm) at 28 C.The following media were used to evaluate LPs production: Luria-Bertani (LB) broth; Cooper's broth (Al-Ajlani et al., 2007), two different brewery wastewaters (BW) obtained from beer production based on barley malt and wheat malt (signed BW#4 and BW#6, respectively), 2% beet molasses (M), apple peels extract (APE) supplemented with 0.25% of yeast extract (YE) or peptone (P) and the carrot peels extract (CPE) with the same supplements. Supernatants obtained by centrifugation (10.000g, 20 min) of 96 h cultures were used for LPs isolation by the method based on the QuEChERS technique (Siewiera et al., 2015; Paraszkiewicz et al., 2017). To obtain the vegetable extracts, proper quantities of carrot and apple peels were boiled for 30 min. After centrifugation, the remaining solid particles were removed by filtration. Distilled water was added to the extracts to a final volume of 1000 ml. All media (with pH adjusted to 7) were sterilized before being used. 2.2. Lipopeptides isolation and mass spectrometry analysis Supernatants obtained by centrifugation (10.000g, 20 min) of 96 h cultures were used for LPs isolation according to the method described by Płaza et al. (2015). LP concentrations in culture supernatants as well as their structures were determined by liquid chromatography e mass spectrometry (LC-MS/MS) and matrixassisted laser desorption time-of-flight mass spectrometry (MALDI-TOF/TOF) techniques (Bernat et al., 2016). Surfactin and iturin standards were obtained from Sigma-Aldrich (Germany) and all other chemicals were purchased from Avantor Performance Materials Poland S.A., Sigma-Aldrich (Germany), Serva (Germany) or Fluka (Germany).
2.1. Culturing of Bacillus strains on different media
2.3. Statistical analysis
The research aim, objectives, and methods are briefly described in Table 1. In detail, two strains of Bacillus subtilis named KP7 and I0 1a were used in this study. KP7 strain was obtained from the collection of the Department of Industrial Microbiology and d Biotechnology, University of Ło z, and I0 -1astrain was supplied by the Institute for Ecology of Industrial Areas in Katowice, Poland. Taxonomic identification and preliminary characterization of strains KP7 and I0 -1a have been described previously by Płaza et al. (2015). The strains were preserved at 70 C in LuriaeBertani (LB) medium supplemented with 20% (v/v) glycerol. Bacterial 24-h-old
All the experiments were performed in triplicates and analysed individually. An average standard deviation was calculated. 3. Results and discussion Data presented in Table 2 reveal that in all growth media used in the study B. subtilis strains were able to synthesise both surfactin and iturin. Moreover, regardless of the used growth medium, KP7 strain produced more surfactin than iturin, while I0 -1a strain overproduced iturin with only traces of surfactin. The same, in all
Table 1 Research aim, objectives and methods of the study. Research principle
Description
Research aim
Determination of the potential of various agro-industrial wastes for surfactin and iturin production. Determination of the structural diversity of the biosurfactants produced by two B. subtilis strains cultured in standard and low-cost media KP7 - surfactin overproducer, producing in LB medium surfactin and iturin in the ratio 17.6: 1; I0 -1a e iturin overproducer, producing in LB medium surfactin and iturin in the ratio 1: 19.8 Two different brewery wastewaters (BW) obtained from beer production based on barley malt and wheat malt (signed BW#4 and BW#6, respectively); 2% beet molasses (signed M); the apple peels extract (APE) and the carrot peels extract (CPE) supplemented with 0.25% of yeast extract (YE) or peptone (P)
Research objectives
Methods
a b
Bacillus subtilis strains Cost-effective culture media containing agroindustrial wastes Control (standard) growth media Biosurfactant isolation Biosurfactant analysis
LuriaeBertani (LB) medium and Cooper's medium Modified QuEChERS technique LC-MS/MSa MALDI-TOF/TOFb
Liquid chromatography e mass spectrometry. Matrix-assisted laser desorption time-of-flight mass spectrometry.
K. Paraszkiewicz et al. / Journal of Environmental Management 209 (2018) 65e70 Table 2 Surfactin production by B. subtilis strains. Growth media Standard media Media prepared from industrial wastewaters Media prepared from vegetable extracts
LB Cooper's BW (#4) BW (#6) M APEþ YE APEþ P CPEþ YE CPEþ P
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Table 4 The surfactin/iturin ratio for tested B. subtilis strains. B. subtilis KP7
B. subtilis I0 -1a
Growth media
100.3 ± 9.18 205.4 ± 21.39 6.6 ± 0.69 9.9 ± 9.18 77.9 ± 0.87 95.2 ± 7.47 104.4 ± 12.6 140.6 ± 8.45 123.6 ± 9.1
3.7 ± 0.32 7.5 ± 0.69 2.3 ± 0.21 1.8 ± 0.19 10.4 ± 1.32 6.3 ± 0.64 7.6 ± 0.69 7.5 ± 0.84 13.9 ± 10.2
Standard media Media prepared from industrial wastewaters Media prepared from vegetable extracts
LB Cooper's BW (#4) BW (#6) M APEþ YE APEþ P CPEþ YE CPEþ P
B. subtilis KP7
B. subtilis I0 -1a
(17.6): (16.6): (16.5): (16.5): (21.6): (52.9): (30.7): (43.9): (49.4):
(1): (1): (1): (1): (1): (1): (1): (1): (1):
(1) (1) (1) (1) (1) (1) (1) (1) (1)
(19.8) (3.1) (6.4) (19.7) (10.9) (27.2) (35.4) (30.1) (30.8)
Luria-Bertani (LB) medium; brewery wastewaters from beer production based on barley malt (BW#4) and wheat malt (BW#6); 2% beet molasses (M); apple peels extract (APE); carrot peels extract (CPE); 0.25% of yeast extract (YE); 0.25% peptone (P).
tested media both surfactin and iturin co-producers retained their individual LP production signature. The literature presents that the medium composition is believed to be an important factor affecting both the production level and the structural diversity of synthesized LPs (Al-Ajlani et al., 2007; Li et al., 2008; Bacon et al., 2012). The above findings are in agreement with data presented in this report. Surfactin production calculated for samples of KP7 strain varied from 6.6 to 205.4 mg L1 (Table 2) while the secretion of iturin by I0 -1a strain was in the range 16.6e428.7 mg L1 (Table 3). The highest surfactin concentration (205.4 mg L1) was obtained in the culture of KP7 strain conducted in Cooper's medium. However, in medium prepared from carrot peels with 0.5% yeast extract (CPEþYE) surfactin production achieved by KP7 strain was higher than in LB medium (140.6 mg L1 and 100.3 mg L1, respectively). The most effective media for iturin production by the strain I0 -1a were CPE and APE supplemented with peptone (428.7 and 269.5 mg L1, respectively). Interestingly, none of the tested media increased the production of iturin by KP7 strain. Some reports published recently demonstrated that various bacilli strains when grown on media prepared from agro-industrial wastes were capable of efficient surfactin production. For example, CagriMehmetoglu et al. (2012) demonstrated that B. subtilis ATCC 6633 produced from 180 to 290 mg L1 when grown in media prepared from rehydrated whey powder. In medium containing glycerol as a main carbon source the production of surfactin by B. subtilis LAMI 009 and LAMI 005 reached 441.1 and 267.6 mg L1, respectively (Sousa et al., 2012). Ali et al. (2014) reported that strain Bacillus RMB7 (exhibiting plant growth promoting potential) produced iturin A in LB culture up to 25 mg L1. Kumar et al. (2015) established that sunflower oil cake favoured high iturin A secretion by rhizospheric isolate of B. amyloliquefaciens up to 276 mg L1. The above mentioned data might suggest that used in our study B. subtilis I0 -1a could be considered as an efficient iturin producer. The surfactin/iturin ratio calculated for KP7 samples (surfactin overproducer) has increased approximately 3-fold in cultures carried out on media prepared from vegetable extracts (all variants of APE and CPE media) as compared with the data obtained from all other cultures (Table 4). Interestingly, a similar relationship was
Table 3 Iturin production by B. subtilis strains. Growth media Standard media Media prepared from industrial wastewaters Media prepared from vegetable extracts
LB Cooper's BW (#4) BW (#6) M APEþ YE APEþ P CPEþ YE CPEþ P
B. subtilis KP7
B. subtilis I0 -1a
5.7 ± 0.43 12.4 ± 2.0 0.4 ± 0.03 0.6 ± 0.06 3.6 ± 0.3 1.8 ± 0.24 3.5 ± 0.17 3.2 ± 0.4 2.5 ± 0.28
73.3 ± 5.86 23.3 ± 1.95 14.6 ± 1.32 35.5 ± 3.11 113.1 ± 9.89 171.2 ± 19.75 269.5 ± 23.34 225.9 ± 25.3 428.7 ± 43.8
observed in samples of I0 -1a strain: in all media based on vegetable extracts cells of B. subtilis I0 -1a (iturin overproducer) increased the production of iturin which resulted in the significant growth of the iturin/surfactin ratio (Table 3). The above data might have suggested that some components of the used vegetable extracts could stimulate the secretion of the LP biosurfactant which tends to be overproduced. Qualitative analysis by MALDI TOF/TOF revealed that KP7 produced two isoforms of surfactin. Fragment ions at m/z 1030 / 917/804 / 689/590 revealed the loss of Leu-Leu-AspVal from the C terminus (Fig. 1S). Furthermore, another typical set of yþ fragment ions at m/z 707 / 594/481 / 382/267 suggested the loss of the Leu-Leu-Val-Asp in the middle of the peptide chain. Therefore the main surfactin isoform (synthesized by both strains in all studied media) was surfactin possessing circular GlueLeueLeu-Val-Asp-Leu-Leu peptide and the b-OH fatty acid. Interestingly, when KP7 strain was growing in the media prepared from vegetable extracts also surfactin isoform with substitution of Leu to Val at position 7 of the circular peptide was produced. Four homologues of surfactin (from C13 to C16; sodiated molecules m/ z1016, 1030, 1044 and 1058 were found in the examined cultures (Fig. 2AS) with the domination of variant C14 ranging between 5565% and 70e85% in samples of KP7 and I0 -1a strains, respectively (Fig. 1). Most often surfactins are synthetized as mixtures of three or four homoloques with acyl chain length in the range C13 - C15 or C13 e C16 (Pecci et al., 2010; Płaza et al., 2015). The MALDI-TOF/TOF MS of extracts of I0 -1a strain showed dominated set of ions m/z 1051.5, 1065.6, 1079.5 and 1093.5 (Fig. 2BS). The molecules differed in their masses by 14 suggesting them to be a members of the same family. Two additional sets of m/ z ions (with corresponding mass difference of M-22 and Mþ39 Da) were also detected which were putatively assigned as iturins (m/z 1029.5, 1043.5 and 1057.5 and 1071.5) and their potassium adducts (m/z 1067.5, 1081.5, 1095.5 and 1109.5). The appearance of sodium and potassium adducts along with their protonated species is a common feature in mass spectral studies of lipopeptides (Pathak and Keharia, 2014). According to the obtained results, it seems that the dominated ions were sodium adducts. Each of these adducts was subjected to the fragment ions analysis. The lower-mass region of the MS/MS spectra indicated peaks corresponding to the immonium ions of the individual constituent amino acids, namely Ser (m/z 60), Pro (m/z 70), Gln (m/z 84), Asn (m/z 87) and Tyr (m/z 136). Thus, the linear acylium ions of iturin could be Pro-Asn-SerbAA-Asn-Tyr-Asn-Gln-COþ. Taken together, these results confirmed that the compound belongs to Iturin A family. MALDI TOF/TOF analysis revealed that both strains produced iturin A. Among four iturin homologues (C13-16) produced by I0 -1a strain, the highest relative contents of C16 variant (70e80%) were calculated for samples obtained from APEþP and CPEþP media (Fig. 2).
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Fig. 1. Relative contents of surfactin homologes produced by B. subtilis KP7 and I0 -1a strains in media prepared from apple peels extract (APE) and carrot peels extract (CPE) supplemented with 0.25% of yeast extract (YE) or 0.25% of peptone (P). Due to a very low abundance (below 3%) the homologue C16 has been not included in the presented calculation.
Fig. 2. Relative contents of iturin A homologes produced by B. subtilis I0 -1a strain in media prepared from apple peels extract (APE) and carrot peels extract (CPE) supplemented with 0.25% of yeast extract (YE) or 0.25% of peptone (P).
On the basis of amino acids variation in the peptide moiety, iturin isomers are classified as iturins A, C, D or E). Moreover iturin A belongs to one of the most frequently detected iturin analogs (Pathak and Keharia, 2014; Cochrane and Vederas, 2014). With the exception of iturin C which does not exhibit the antibiotic activity, other iturin isomers reveal increasing biological activity with a growing number of carbon atoms in the fatty acid chain. In conclusion, the data obtained in this research revealed a great potential of I0 -1a strain due to its ability for very effective production of iturin A during the growth on media prepared from vegetable extracts.
The potential applications of biosurfactants in various industries has significantly increased in recent years; however, economical large-scale production of biosurfactants remains a challenge (Dubey et al., 2012). Preliminary work by Płaza et al. (2011) confirmed that three Bacillus spp. were capable of growing and producing biosurfactants in media composed of brewery effluents, molasses and fruit as well as vegetable decoction from a processing factory. More recently, simultaneous production of detergent stable enzymes and biosurfactant by Bacillus subtilis PF1 growing in the single medium containing cost effective agroindustrial wastes (namely feather meal, potato peel and rape seed cake) was reported
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~ ez-Ocampo et al. (2017) used waste by Bhangea et al. (2016). Yan cooking oil and coffee wastewater as carbon sources for the production of biosurfactants exhibiting the ability to be potentially exploited for the treatment of soil contaminated with insoluble compounds (pesticides or oil hydrocarbons). Various unconventional agroindustrial wastes, (molasses, orange peels extract, bagasse extract, banana peels extract and potato peels extract) were used for the production of biosurfactant from the indigenously isolated Bacillus subtilis ANR 88, examined further towards its possible application in nanoparticle synthesis (Rane et al., 2017). Previously, B. subtilis I0 -1a and KP7 grown on LB medium were described as producers of surfactin, iturin and fengycin mixture (Płaza et al., 2015). Nevertheless, fengycin production by I0 -1a and KP7 strains still requires further study. It was also found that LPs extracted from bacterial cultures had a strong antimicrobial effect on uropathogenic bacteria, including effects on planktonic growth, and biofilm formation and removal (Moryl et al., 2015). Moreover, Bernat et al. (2016) elucidated the structure of LPs produced by B. subtilis strain I0 -1a grown on the traditional microbiological LuriaeBertani (LB) medium. B. subtilis strain I0 -1a was also found to produce a mixture of surfactin homologues (C13-C16). In addition, two iturin A homologues, C14 and C15 were observed, of which C15 was dominant. The obtained results reveal that available renewable natural resources could be used for effective iturin A production by the strain B. subtilis I0 -1a. 4. Conclusions The wastes from various sectors of agro-industry containing many organic compounds could be utilized as substrates for biosurfactant production, which resulted in a double benefit i. e., reduction of the wastes as well as production of valuable residues like biosurfactant. As estimated the use of low-cost raw material is a factor that accounts for a 10e30% of the overall cost of biosurfactant production and can solve various industrial waste management problems. The promising future of biosurfactants depends on the use of a large and low cost raw materials to achieve high biosurfactants yields. In this context, our paper addressed the application of various agro-industrial wastes for biosurfactant production and for the determination of their chemical structure. In detail, the potential of agro-industrial wastes (two different brewery wastewaters, 2% beet molasses, apple peels extract supplemented with 0.25% of yeast extract or 0.25% peptone, and similarly supplemented carrot peels extract) as low-cost culture media substrates for biosurfactant production by two B. subtilis strains was investigated. As compared to data obtained for Luria-Bertani broth (a standard medium used as a control) the production of surfactin by surfactin overproducer B. subtilis KP7 grown in carrot peels extract supplemented with east extract was 140% over the control, whereas the production of iturin by iturin overproducer B. subtilis I0 -1a cultured in carrot peels extract supplemented with peptone was as high as 585% over the control produced. We found that agricultural substrates with different compositions used as bacterial growth media influence the type of biosurfactant produced. The application of wastes in biosurfactant production is still at an early stage. However, the results published in this area are promising. Conflict of interest The authors declare no conflict of interest. Acknowledgements This paper was prepared in connection with work conducted under project (no 2013/09/B/NZ9/01759) that was sponsored by the
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